Organoid-Based Transcriptomics Indicate IFIT-Associated Immune Modulation during Cryptotanshinone Treatment in Bladder Cancer

Bladder cancer (BC), particularly muscle-invasive or metastatic disease, remains a major clinical challenge despite advances in immunotherapy. In this study, five candidate small molecules were screened, and cryptotanshinone (CTS) was identified as the most promising compound. Using BC cell lines and bladder tumor organoids, we evaluated the effects of CTS on proliferation, migration, apoptosis, and organoid growth. Transcriptomic sequencing of bladder tumor organoids, combined with protein–protein interaction analysis and public databases (TCGA-BLCA, TIMER, and TISIDB), was used to characterize CTS-associated molecular features. Integrative analyses identified IFIT1, IFIT2, and IFIT3 as representative interferon-stimulated candidate genes associated with BC progression, prognosis, immune infiltration, and PD-1/PD-L1 expression. These findings were supported by qRT-PCR and Western blot analyses showing reduced IFIT1/2/3 expression at both mRNA and protein levels following CTS treatment. Molecular docking suggested potential associations between CTS and interferon-related signaling proteins. Consistently, CTS reduced TBK1 and STAT1 phosphorylation, providing preliminary experimental support for its association with interferon-related signaling modulation. Collectively, CTS exerts growth-inhibitory effects and is associated with tumor cell-intrinsic interferon-related transcriptional and signaling changes in BC models, providing a basis for further investigation as a small-molecule candidate for BC. • CTS was identified as the most potent inhibitor among five small-molecule candidates. • CTS suppresses bladder cancer cell proliferation, migration, apoptosis resistance, and organoid growth. • Transcriptomic profiling identified IFIT1, IFIT2, and IFIT3 as CTS-responsive immune-related genes. • CTS downregulated IFIT1/2/3 expression at the mRNA level in bladder cancer organoids and at the protein level in bladder cancer cells. • CTS may attenuate interferon-related signaling activity through modulation of TBK1 and STAT1 phosphorylation.